This application is the national phase under 35 U.S.C. xc2xa7371 of prior PCT International Application No., PCT/JP96/03873, which has an International filing date of Dec. 27, 1996, which designated the United States of America, the entire contents of which are hereby incorporated by reference.
The present invention relates to a method for producing plants with novel properties, more specifically, a method for producing temperature-tolerant plants which are highly resistant to environmental stress.
Many organisms adapt themselves to severe environmental stress by synthesizing and accumulating a specific compound called xe2x80x9ccompatible solutexe2x80x9d in their cytoplasm to protect themselves against such stress. Environmental stress to which organisms have been shown to adapt themselves by such a mechanism include salts (Imhoff et al., FEMS Microbiol. Rev. 39:57-66, 1986; Mackay et al., J. Gen. Microbiol. 130:2177-2191, 1984; Rhodes and Hanson, Annu. Rev. Plant Physiol. Plant Mol. Biol. 44:357-384, 1993), dehydration (Yancy et al., Science 217:1214-1222, 1982) and low temperatures (Ko et al., J. Bacteriol. 176:426-431, 1994).
Among those compatible solutes, glycine betaine (hereinafter referred to as betaine) is widely distributed in higher plants (Robinson and Jones, Aust. J. Plant Physiol. 13:659-668, 1986), bacteria (Csonka, Microbiol. Rev. 53:121-147, 1989) and animals (Garcia-Perez and Burg, Physiol. Rev. 71:1081-1115, 1991; Lever et al., Biochim. Biophys. Acta. 1200:259-264, 1994). As shown in FIG. 1, betaine is a bipolar compound having a positive charge and a negative charge in its molecules (Rhodes and Hanson, Annu. Rev. Plant Physiol. Plant Mol. Biol. 44:357-384, 1993). A long discussion regarding the physiological functions of betaine has suggested that betaine may protect cells by maintaining an osmotic balance with the environment (Robinson and Jones, Aust. J. Plant Physiol. 13:659-668, 1986) and that betaine may stabilize higher-order structures of proteins (Bernard et al., Acad. Sci. 111, 307:99-104, 1988; Papageorgiou and Murata, Phtosynth. Res. 44:243-252, 1995). However, betaine is not exclusively synthesized in cells under salt stress or dehydration stress. Thus, it could not be concluded that betaine has a direct effect on the protection of cells against such stress.
In Escherichia coli and spinach (Spinacia oleracea), betaine is biosynthesized from choline via two steps of oxidation as shown in FIG. 1. On the other hand, choline oxidase obtained from the gram-positive soil bacterium Arthrobacter globiformis can oxidize choline to betaine in one-step oxidation reaction (Ikuta, S. et al., J. Biochem. 82:1741-1749, 1977).
In an attempt to study a direct effect of betaine, we isolated the coda gene encoding a novel choline oxidase which catalyzes oxidation from choline to betaine (Japanese Society of Plant Physiologist, Annual meeting of 1994, the 34th Symposium held Mar. 28-30, 1994) and integrated it into cells of the cyanobacterium strain Synechococcus PCC7942 and brassicaceous plants, thus succeeded in obtaining salt-tolerant and/or osmotolerant plants (Japanese Patent Application No. 106819/95). This confirmed that betaine functions to protect organisms against salt stress.
However, no report has shown that betaine confers temperature tolerance on plants or bacteria.
It is an object of the present invention to produce plants that are tolerant to environmental changes such as high temperatures or low temperatures by gene recombination techniques.
As a result of careful study to solve the above problems, we succeeded in obtaining temperature-tolerant plants by integrating and expressing a gene encoding choline oxidase in cyanobacteria, brassicaceous plants and gramineous plants.
Accordingly, the present invention provides a method for producing temperature-tolerant plants, which comprises transforming a plant with a recombinant vector carrying a gene encoding choline oxidase.
The present invention also provides temperature-tolerant plants produced by said method or progenies thereof having the same properties.